ABSTRACT The goals of this project are to use genomic approaches to identify, in the human genome, powerful chromatin insulators and powerful erythroid specific enhancers. Use of powerful insulators in therapeutic gene therapy vectors will increase safety by decreasing the risks of vector-related genotoxicity. Availability of powerful erythroid specific enhancers will increase the efficacy of gene therapy in patients with severe thalassemia. With the studies of the first specific aim we will discover powerful chromatin insulators in the human genome using the novel genomic screening approaches we have developed. Special emphasis will be given in identifying powerful barrier insulators which also possess powerful enhancer blocking activity. Such insulators are expected to improve both the safety and the efficacy of integrating gene therapy vectors. We will determine the structural characteristics of the new insulators and we will investigate the structural basis of the variation in their potency. In specific aim 2 we will discover new powerful erythroid specific enhancers in the human genome using molecular and informatics methods. We will verify the erythroid lineage specificity of the new enhancers, we will characterize them and will use them to identify the molecular features that determine erythroid lineage specificity. With the studies of Specific Aim 3 we will produce a new generation of globin gene therapy vectors. The most powerful insulators of Specific aim 1 will be used to determine the optimal sizes, the highest titers and the highest protection from genotoxicity required for the gene therapy vectors. The most powerful enhancers of Specific Aim 2 or enhancer cassettes will be used for quantitative assessments of their potency in supporting globin gene expression. The best insulators and enhancers will be incorporated in globin gene vectors and their therapeutic potency will be assessed ex vivo in the erythroid cells of thalassemic patients and in vivo in thalassemic mice.